Hdm-2 targeting compositions cause tumor cell necrosis rather than apoptosis of cancer cells

ABSTRACT

An aspect of the invention provides a method of selectively necrosing cells, comprising: providing a plurality cells, including at least one cancer cell and at least one non-cancerous cell; and administering to the cells a compound, including an HDM-2 targeting component and a cytotoxic component attached to the HDM-2 targeting component, wherein said compound comprises a membrane-active form.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/470,488, filed Aug. 27, 2014, which is a continuation ofU.S. patent application Ser. No. 13/122,256, filed Sep. 14, 2011, whichis the national stage filing of International Patent Application No.PCT/US2009/059380, filed Oct. 2, 2009, which claims the benefit of andpriority to U.S. Provisional Patent Application Ser. No. 61/102,590filed on Oct. 3, 2008, the contents of which are incorporated herein byreference in their entirety.

FUNDING STATEMENT

This invention was made with government support by a Veteran'sAdministration Grant (WBB) and The American College of Surgeon's FacultyResearch Fellowship Award 2007-2009 (WBB).

FIELD OF THE INVENTION

The invention relates to methods of effectively treating various formsof cancer and screening candidate cancer treatments and compounds.Specifically, the present invention is directed to the use of novelcompounds and methods to treat cancer and non-cancerous cells and causenecrosis only to cancer cells.

RELATED ART

Cancer treatments which target the p53 protein within the cancer cellshave been developed recently. However, some types of cancer cells do nothave p53, while others exhibit p53 in a mutated, and/or inactive form.Thus, these p53 targeting cancer treatments are limited since they donot cause cell death in these types of cancer cells. Thus, p53-targetingcancer treatments are ineffective at treating various types of cancer.

SUMMARY OF THE INVENTION

The embodiments of the present invention are directed to the surprisingdiscovery that cancer cells have approximately several times as muchHDM-2 in their cellular, mitochondrial, and nuclear membranes thannon-cancerous cells. Thus, HDM-2 targeting compounds are successfulcancer treatments, causing necrosis to cancer cells while leavingadjacent non-cancerous cells unaffected. HDM-2 targeting treatments thusrepresent a body of wide-acting cancer treatments that are moreeffective than the current, limited p53-targeting treatments.

An aspect of the invention provides a method of selectively necrosingcancer cells, but not untransformed or normal cells. The method includesthe steps of: administering to the cells a compound, wherein thecompound includes an HDM-2 targeting component and a cytotoxiccomponent, where the cytotoxic component may be attached to said HDM-2targeting component such that the compound comprises a membrane-activeform. An example of an HDM-2 targeting component may include, forexample, one or more small molecules, a peptide, a protein, aglycoprotein, an antibody (including whole and fragment antibodies), andcombinations thereof. Examples of a cytotoxic component may include: amembrane resident peptide (MRP), a toxin, a drug, a radionuclide, anantibody (including whole and/or fragment), and combinations thereof, asmay be desired. One or more of the cytotoxic components, including thetoxin, drug, radionuclide, antibodies, and combinations thereof, may beknown and/or used in the art, for their cytotoxic affects to cells,optionally, cancer cells.

Optionally, the HDM-2 targeting may be a peptide. Where the HDM-2targeting component and the MRP are both peptides, the MRP is preferablyattached to the carboxyl terminal end of the peptide.

Optionally, the method may further include the step of observing therelease (from the cancer cell) of an increased LDH amount as compared toan initial LDH amount from the cancer cell, observing necrosis in thecancer cells, and/or observing a non-response in the normal cell. Thenon-response of a normal cell may indicate that the normal cell isunaffected by the cancer treatment.

Another aspect of the present invention provides a method of causingmembranolysis in at least one cancer cell. The method includes the stepof administering to at least one cancer cell a compound including anHDM-2 targeting component and a MRP, the MRP attached to the HDM-2targeting component.

Optionally, the method may further include, for example, observingmembranolysis in the cancer cell by microscopy. Observing necrosis ofthe cancer cell may also be included as a step in the present invention.

Still another aspect of the present invention provides a method oftreating cancer in a subject (or patient) in need thereof. The methodincludes the steps of administering to the subject in need thereof atherapeutically effective amount of a compound having an HDM-2 targetingcomponent and a MRP, the MRP attached to the HDM-2 targeting component.The subject may include, for example, mammals including dogs, cats,chimpanzees, and rats. Optionally, the method may further include thestep of correlating a result thereof of the administration step.

Still yet another aspect of the present invention provides a method ofscreening candidate cancer treatments. The method may include the stepsof: providing a plurality of cancerous cells; administering a candidatecancer treatment to the plurality of cancerous cells; and measuring thelevel of LDH released from said cells. LDH is measurable in the cellmedium, once it is released from the cells.

Optionally, the method includes administering the candidate cancertreatment which may include a compound including an HDM-2 targetingcomponent and an MRP. As the method employs screening compounds fortheir potential abilities as (1) binding affinity for HDM-2 and (2)membrane transport character, one or both of these characteristics maybe desired in various drug candidates that are screened with the presentmethod. The screening process, may aid in identifying components thatact within the desired parameters and with the preferablecharacteristics as effective cancer treatments. Additionally desirablecharacteristics of cancer treatment, including causation ofmembranolysis and ultimately, cancer cell necrosis may be observed orotherwise measured after each candidate compound is administered. Thus,the efficacy of each candidate may be screened.

Optionally, the method may also contain the steps of observing the cellsfor LDH, and/or correlating the level of LDH in the cellular medium to astandard. Thus, necrosis, and the level thereof may be identified foreach candidate, as it may correlate to the level and/or amount of LDHreleased for a given sample.

Still yet another aspect of the present invention provides a method ofselectively necrosing cancer cells, including the steps of: providing atleast one cancer cell and at least one non-cancerous cell; andcontacting the cells with a compound, where the compound includes anHDM-2 targeting compound having an MRP attached thereto, wherein thecompound binds to a cancer cell membrane and configures to a membraneactive form, binding to the cancer cell membrane. This binding site ispreferably at a site of HDM-2, and results in trans-membrane poreformation in the cancer cell membrane.

The method optionally includes the steps of measuring a level of LDHfrom the cancer cell, observing necrosis of the cancer cell, and/orobserving a non-result in the non-cancerous cell as a result of theadministering step.

A further aspect of the present invention provides a method ofidentifying cancer cells from a plurality of cells, including: providinga plurality of cells, wherein at least one of the cells is a candidatecancer cell; administering to the plurality of cells an HDM-2recognition compound; and observing the plurality of cells for the HDM-2recognition compound to bind to a cell membrane of at least one of thecells, where binding to the cell membrane is indicative of a cancerouscell.

Optionally, the method may include the step of fluorescing the HDM-2recognition molecule with an observable agent. The observable agent mayinclude, for example, various known dyes, enzyme-substrate combinations,radiopaque materials, fluorescing agents, and combinations thereof. Theobservable agents may be visually observable, observable with filteredlight through various scopes, and/or indentified through X-ray and orother medical instrumentation photography. With HDM-2 targetingcompounds used in conjunction with observable agents, cancerous areasmay be indentified, topographically mapped, and better understood thanwith previous cancer identification and visualization techniques.

The method may further include the step of classifying an identifiedcancer cell as a type of cancer. This may be done in vivo, as part of adiagnostic for cancer. Alternatively, the fluorescent-labeled MRPattached to an HDM-2 targeting component or HDM-2 recognition agent maybe administered to a candidate surgical area. Such a use may providederivative identifiers to a surgeon of the highly cancerous tissue, lesscancerous tissue, and non-cancerous tissue for surgical removal and/orintervention purposes. As such, the embodiments and features of thepresent invention may provide a great aid in surgical pathology, helpingpathologists to distinguish cancer from non-cancer.

The description of the elements and features of the present inventionand equivalents thereof may be better understood through a study of thefollowing drawings, description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is experimental data which illustrates that PNC-28 is cytotoxicto MiaPaCa-2 cells. Panel A shows untreated MiaPaCa-2 cells incubatedfor 24 h. Panel B represents MiaPaCa-2 cells treated for 24 h with 75μmol/ml of PNC-28. Panel C shows MiaPaCa-2 cells treated with 75 μmol/mlPNC-29 negative control peptide for 48 h.

FIG. 2 is a chart of experimental data which illustrates that PNC-28(diamonds) is cytotoxic to MiaPaCa-2 cells in a dose-dependent mannerover a dose range of 0 μmol/ml up to 160 μmol/ml. Specifically, a dosageof 20 μmol/ml caused roughly 35% cell death, while a dosage of 80μmol/ml caused over 80% cell death at 48 h. The effect of the negativecontrol PNC-29 (squares) is also shown. The effective dose range forPNC-28 at 48 h from 20 to 80 μmol/ml is strongly statisticallysignificant (P<0.001).

FIG. 3 is a chart of experimental data which illustrates that thecarboxyl terminal attached MRP to residues 17-26 (PNC-28) is requiredfor cytotoxicity to cancer cells. The chart depicts the number ofremaining cells after 48 hours have passed, shown as a function oftreatments administered. MiaPaCa-2 cell death following treatment withno peptide (condition 1), 75 μmol/ml of PNC-28 (condition 3), PNC-26(condition 2), and negative control PNC-29 (condition 4) after 48 h oftreatment.

FIG. 4A is a chart of experimental data which illustrates that PNC-28induces cellular death by necrosis in MiaPaCa-2 cells. LDH activity(measured as absorbance/optical density at 492 nm) was recorded forMiaPaCa-2 cells incubated with 25 μmol/ml of PNC-28 (condition 2), nopeptide (condition 3), and PNC-29 (condition 4) at 24 h. Maximal LDHrelease is shown after treatment with known lysis buffer (condition 1).

FIG. 4B is experimental data which illustrates comparative electronmicrographs of MiaPaCa-2 cells that were untreated (right panel) versustreated (left panel) with 25 mol/ml PNC-28 for 15 min. The arrows in theleft panel point to gaps or holes in the cell membrane induced byPNC-28.

FIG. 4C is a chart of experimental data which illustrates thatPNC-28-induced cell death not caused by apoptosis. Caspase 3 activity,an apoptosis indicator, was recorded for MiaPaCa-2 cells incubated with25 μmol/ml of PNC-28 (condition 2), no peptide (condition 3), and PNC-29(condition 4) at 24 h. Maximal caspase release is shown after treatmentwith TNF-α (condition 1) known to induce apoptosis. Caspase 3 activityis measured by luminescence (UL), as shown on the y-axis.

FIG. 4D is a chart of experimental data which illustrates that PNC-28induces cell death by causing tumor cell necrosis, and not apoptosis,over its entire effective concentration range. At each dose, both LDHand caspase activity were measured after incubation with PNC-28 after 24h. For the points on the abscissa, two numbers are separated by a dash.The first number refers to the concentration of peptide; the secondnumber refers to the particular peptide, e.g., “28” refers to PNC-28,while “29” refers to the negative control, PNC-29. Optical Density isshown as a function of treatment.

FIG. 5 is experimental data in the form of a chart which illustratescell death (number of dead cells divided by total cell count) asmeasured by trypan blue dye exclusion in MiaPaCa-2 and BMRPA1 cellstransfected with either p53 17-26-V or control p53 17 scrm-V plasmid, 48h post-transfection. Condition 1: MiaPaCa-2 cells transfected with p5317-26-V (black); condition 2: Mia-PaCa-2 cells transfected with p5317-26-scrm-V (white); condition 3: BMRPA1 cells transfected with p5317-26-V (black); condition 4, BMRPA1 cells transfected with p5317-26-scrm-V (white).

FIG. 6 is experimental data which shows the effects of expression of thep53 17-26 peptide, following transfection of its expression vector intoMiaPaCa-2 cells, on expression of p53 and waf^(p21), a cell cycleinhibitor protein induced by activated wild-type p53, as a function oftime, measured by immunoblotting and on caspase activity. Peptideexpression in cells was measured by blotting for the peptide with theanti-p53 monoclonal antibody DO-1 that recognizes the p53 17-26 sequenceexpressed by the plasmid. For comparison, the effects of incubatingPNC-28 with MIA-PaCa-2 cells on induction of these proteins and oncaspase activity are also shown. Intracellular PNC-28 level wasdetermined using the same DO-1 antibody. The left ordinate shows theabsorbance results for the caspase activity assay while the rightordinate shows the band intensity for each Western blot; the actualimmunoblots are shown above each bar graph for the two proteins, p53,waf^(p21) and each peptide (p53 17-26 and PNC-28). The left side of thefigure shows the results for 0 time, after Mia-PaCa-2 cells weretransfected with the plasmid (labeled as “transfection” in the figure)and immediately after PNC-28 was added to the incubation medium (labeledin the figure as “PNC-28.”). The right side of the figure shows theresults after close to 100 percent of the cells were killed by theplasmid-expressed peptide (labeled in the figure as “transfection”) andby PNC-28 (labeled in the figure as “PNC-28”). Actin controls were thesame for all four conditions (not shown).

FIG. 7 is experimental data which illustrates that transfection of p5317-26 vector induces apoptosis in MiaPaCa-2 but not BRMPA1 cells.Confocal microscopy demonstrating green fluorescence followingtransfection of control vectors into MiaPaCa-2 and BMRPA1 cells (PanelsA and C). Annexin V binding to phosphatidyl serine (red staining)detected in p53 17-26-V-treated MiaPaCa-2 cells (Panel B) but not in17-26-V-treated BMRPA1 cells (D).

FIG. 8A is a chart of experimental data which illustrates that p53 17-26induces cellular death by apoptosis. Caspase 3, 7 activity recorded(luminescence as labeled on the ordinate) for MiaPaCa-2 cellstransfected with p53 17-26-V (condition 3), empty vector (condition 2),PNC-28 (condition 4), and PNC-29 (condition 5). Maximal caspase releaseis shown after treatment with TNF-α condition 1).

FIG. 8B is a chart of experimental data which illustrates that p53 17-26induced cell death does not cause necrosis. LDH activity recorded forMiaPaCa-2 cells transfected with p53 17-26-V (condition 3), empty vector(condition 2), PNC-28 (condition 4), and PNC-29 (condition 5). MaximalLDH release is shown after treatment with lysis known buffer (condition1).

FIG. 9 depicts a table summarizing the efficiency of transfection ofplasmids into MIA-PaCa-2 and BMRPA1 cells.

DETAILED DESCRIPTION OF THE INVENTION

p53 is the gene that is most commonly disrupted in cancer. p53 acts asthe guardian of the genome, as it guards against copying of the DNA. Itwas previously established that p53 gene within the cells was a targettreatment for cancer. However, p53 targeting treatment in cancer cellshas various problems associated with it that limits the use of p53targeting treatments. For example, not all cancers exhibit p53 in thecell. Targeting treatments for these types of cancers would not work, asthere is no p53 for the targeting compounds to bind to. Also, somecancers exhibit a mutated form of p53, which is inactive. As the p53 isinactive in these cancers, targeting compounds also do not work forthese cancers. Thus, p53 dependent treatment mechanisms are ineffectiveagainst these types of cancer.

The materials and methods of the present invention provide novel methodsof treatment that are directed to a common characteristic in manyvarious forms of cancer. Such materials and methods may be used as atargeted treatment to many various forms of cancer, which, up until now,may have very different and less effective and predictable treatmentoptions and avenues. These new methods, materials, and screening methodsprovide effective treatments, screening methods for additional noveldrug candidates, and other benefits and advantages over the currentmedical technology.

As used herein, cancer includes any disease or disorder associated withuncontrolled cellular proliferation, survival, growth, or motility.Cancers that may be treated or prevented by the present inventioninclude any cancer whose cells have increased expression of HDM-2 intheir plasma membranes. Such cancers may include, for example,pancreatic cancer, breast cancer, colon cancer, gastric cancer, prostatecancer, thyroid cancer, ovarian cancer, endometrial cancer,glioblastoma, astrocytoma, renal carcinoma, lung cancer, sarcoma,including osteogenic sarcoma, mesothelioma, sporadic nonfamilial tumors,lymphoma, and others including hematologic cancers such chronicmyelogenous leukemia. Precancerous conditions, where cells exhibit highamounts of HDM-2 in the plasma membrane, are also included as treatablewith the compositions and methods of the present invention.

The present invention is directed to the surprising discovery of theinventors that cancer cell membranes and nuclear membranes have a largeamount of HDM-2 as compared to normal non-cancerous cells. HDM-2 andMDM-2 (human double minute vs. mouse double minute) each have a p53binding domain. HDM-2 and MDM-2 are found in the cell and nuclearmembranes of cancer cells, but not in normal, healthy cells. Thecompounds of the present invention include a HDM-2 targeting componentand a Membrane Resident Peptide (MRP), where the MRP is attached to thecarboxyl terminal end of the HDM-2 targeting component. The MRP mayinclude the residues that are shared by the p53 binding domain (orregion) of HDM-2. As such, when the compound is administered orotherwise contacted to at least one cancer cell, the HDM-2 targetingcomponent, which has a p53 binding domain, may bind to the HDM-2 in thecancer cell membrane. The presence of the MRP on the end allows thecompound to become membrane-active and to form well-defined pores in thecell membrane (membranolysis), which allow for extrusion of theintracellular contents and compromise the integrity of the cell. Poresin the cancer cell membrane are formed as an immediate result ofadministration of the compound. After the pores are formed, cellnecrosis, or cell death, results within a short time frame. Thus, cancertreatments having an HDM-2 targeting component and an MRP are effectivetreatments for cancer.

Cancer cells have approximately five times as much HDM-2 as normal cellsin their cellular membranes and nuclear membranes. Though the cancersmay not exhibit similar characteristics or typical treatment avenues, asmany cancers each have HDM-2 in their cell membranes, these cancers arelikewise susceptible to the methods of treatment of the presentinvention. Cancers that have been identified as having a large amount ofHDM-2 in the cell membrane include, for example: MIA-PaCa-2 humanpancreatic cancer cells, MCF-7 human breast cancer cells, B16 mousemelanoma, and a human melanoma cell line A2025. The compounds which haveboth an HDM-2 targeting component and an MRP thus have a high affinityfor cancer cells, and will thus only bind to and cause necrosis ofcancer cells when administered to a combination of cancer cells andnormal, healthy cells.

The present inventors have discovered methods and uses of the compoundscontaining an HDM-2 binding domain having a MRP attached at its end,where the compound is specifically designed to target cancer cells andnot target normal, healthy cells of a sample. Where both the HDM-2targeting component and the MRP are peptides, the MRP is desirablyattached to the carboxyl terminal end of the HDM-2 targeting component.

Thus, these methods may be used to treat a sample of cells containingboth healthy, normal cells and cancer cells. Such samples would includecell lines, tissue samples, tumors, and/or a subject having cancer inneed of treatment. As the methods of treatment do not cause cell deathof normal cells, these methods of treatment are focused on the cancercells, irrespective of the mode of administration to the cell sample.Thus, these methods of treatment may be used for tumors or cancers thatare widespread, inoperable, or otherwise not easily treated withconventional means or combination therapies.

The methods of the present invention kill cancer cells by necrosis.Necrosis is induced by the combined action of the compound, which actsboth to bind to HDM-2 and to form pores on the cellular membrane. Thepores ultimately cause the cell membrane to lose its integrity such thatintracellular contents leak from the cell, and the cell undergoesnecrosis.

The present invention provides methods of using HDM-2 targeting cancercompounds which correspond to all or a portion of amino acid residues12-26 of human p53 protein. When fused to a MRP, the peptides are lethalto malignant or transformed cells. The subject cancer treatmentcompounds may be useful in treating neoplastic disease in an animal,preferably a human.

The compounds of the present invention may include, for example, PNC-27and PNC-28. Additionally, one or more compounds may be used, where acompound may have an HDM-2 targeting component. The HDM-2 targetingcomponents may be, for example, the residues of p53 which bind to HDM-2.Further, the compound may include a membrane resident peptide, or, MRP.Both PNC-27 and PNC-28 are examples of p53-derived peptides from thehuman double minute binding domain (HDM-2) that are attached to MRP.These compounds induce tumor cell necrosis of cancer cells, but notnormal cells. The anti-cancer activity and mechanism of PNC-28 (p53aa17-26-MRP) was specifically studied by the inventors of the presentinvention as against human pancreatic cancer, though uses andapplications are included with the various methods of the presentinvention.

The inventors show with the present invention and supportingexperimental examples that the MRP is necessary for this action sinceexpression of the naked p53 sequence without MRP in cancer cells causeswild-type p53-dependent apoptosis, or programmed cell death, not tumorcell necrosis.

Preferably, the MRP includes predominantly positively charged amino acidresidues since a positively charged leader sequence, which may stabilizethe alpha helix of a subject peptide. Examples of MRPs which may beuseful to the HDM-2 targeting compounds of the present invention aredescribed in Futaki, S. et al (2001) Arginine-Rich Peptides, J. Biol.Chem. 276, 5836-5840, and include but are not limited to the followingMRPs in the TABLE 1, below. The MRP may be, for example, peptidesincluded in SEQ ID NO:1, or 9-29. The numbering of the amino acidresidues making up the MRP is indicated parenthetically immediatelyafter the name of the component in most of the examples in most of thesequence listings.

TABLE 1 SEQ ID NO:  Sequence NAME SEQ ID NO: 1 KKWKMRRNQFWVKVQRGMembrane resident peptide (MRP), reverseomer of AntennapediaSEQ ID NO: 2 PPLSQETFSDLWKLL PNC-27 KKWKMRRNQFWVKVQRG SEQ ID NO: 3ETFSDLWKLLKKWKM PNC-28 RRNQFWVKVQRG SEQ ID NO: 4 MPFSTGKRIMLGEKKWKPNC-29 MRRNQFWVKVQRG SEQ ID NO: 5 MPFSTGKRIMLGE peptide fromcytochrome P450 (aka “X13”) SEQ ID NO: 6 TIEDSYRKQVVIDKKWK PNC-7MRRNQFWVKVQRG SEQ ID NO: 7 TIEDSYRKQVVID ras-p21 residues 35-47SEQ ID NO: 8 PPLSQETFSDLWKLL PNC-26, residues 12- 26 of the HDM-2binding domain of p53 SEQ ID NO: 9 YGRKKRRQRRRPPQ HIV-1 TAT(47-60),membrane resident peptide SEQ ID NO: 10 GRKKRRQRRRPPQ D-TAT, membraneresident peptide SEQ ID NO: 11 GAAAAAAAAAPPQ R-TAT G(R)₉PPQ,membrane resident peptide SEQ ID NO: 12 PKKKRKV SV40-NLS, membraneresident peptide SEQ ID NO: 13 KRPAAIKKAGQAKKKK nucleoplasmin-NLS,membrane resident peptide SEQ ID NO: 14 TRQARRNRRRRWRERQRHIV REV (34-50), membrane resident peptide SEQ ID NO: 15 RRRRNRTRRNRRRVRFHV (35-49) coat, membrane resident peptide SEQ ID NO: 16KMTRAQRRAAARRNRWTAR BMV GAG (7-25), membrane resident peptideSEQ ID NO: 17 TRRQRTRRARRNR HTLV-II REX 4-16, membrane resident peptideSEQ ID NO: 18 KLTRAQRRAAARKNKRNTR CCMV GAG (7-25), membrane residentpeptide SEQ ID NO: 19 NAKTRRHERRRKLAIER P22 N (14-30), membrane residentpeptide SEQ ID NO: 20 MDAQTRRRERRAEKQAQWKAAN LAMBDA N(1-22),membrane resident peptide SEQ ID NO: 21 TAKTRYKARRAELIAERRPhi N (12-29), membrane resident peptide SEQ ID NO: 22 TRRNKRNRIQEQLNRKYEAST PRP6 (129- 124), membrane resident peptide SEQ ID NO: 23SQMTRQARRLYV HUMAN U2AF, membrane resident peptide SEQ ID NO: 24KRRIRRERNKMAAA HUMAN C-FOS KSRNRRRELTDT (139-164), membraneresident peptide SEQ ID NO: 25 RIKAERKRMRNRIA HUMAN C-JUN ASKSRKRKLERIAR(252-279), membrane resident peptide SEQ ID NO: 26 KRARNTEAARRSRAYEAST GCN4, RKLQRMKQ membrane resident peptide SEQ ID NO: 27KLALKLALKALKAALKLA Example membrane resident peptide (MRP) SEQ ID NO: 28LLIILRRRIRKQAKAHSK p-vec, membrane resident peptide SEQ ID NO: 29RRRRRRRR (Arg)s or any poly-R from (R)₄-(R)₁₆, membrane resident peptideSEQ ID NO: 30 GCCACCATGG Kozak sequence SEQ ID NO: 31 AGTCGAATTCGCCsense strand sequence ACCATGGAAACAT of cDNA encoding the TTTCAGACCTATGp53 17-26 sequence GAAACTACTTTGA GCGGCCGCAGTC SEQ ID NO: 32 ETFSDLWKLLresidues 17-26 of HDM-2 binding domain of p53

Other MRP materials may also be used. Such sequences are described e.g.,in Scheller et al. (2000) Eur. J. Biochem. 267:6043-6049, and Elmquistet al., (2001) Exp. Cell Res. 269:237-244, the contents of which areincorporated herein by reference in its entirety.

Desirably, the positively charged MRP may include the amino acidsequence: KKWKMRRNQFWVKVQRG (SEQ ID NO: 1), which is related to thereverseomer sequence of the antennapedia sequence. Preferably, the MRPis attached to the carboxyl terminal end of a subject compound (e.g.peptide).

Cell death can occur by either necrosis or apoptosis. p53-targetingtreatments typically cause cell death through apoptosis, while thecompounds and methods of the present invention cause cell death bynecrosis. Necrosis is not genetically controlled, while apoptosis isgenetically controlled. Apoptosis is the deliberate cellular response tospecific environmental and developmental stimuli or programmed celldeath. Cells undergoing apoptosis exhibit cell shrinkage, membraneblebbing, chromatin condensation and fragmentation. Necrosis involvesthe destruction of cytoplasmic organelles and a loss of plasma membraneintegrity. Though apoptosis does not have the inflammation which resultswhen cancer cells die through necrosis, p53 targeting treatments fail totreat those cancers that do not exhibit p53, or, through mutations,exhibit an inactive p53 form that is unresponsive to p53 targetedtreatments. After the DNA damage in the caspase enzyme pathway, thereare a series of events which occur that involve calcium activation andcalpain enzymes which further leads to other cellular changes andregulation of cytoplasmic enzymes. During p53-dependent apoptosis, thereis a sequential expression of annexin V-binding membrane phospho-Serine,Bax waf^(p21), and caspases; these proteins are used as markers forp53-dependent apoptosis.

A major difference between necrosis and apoptosis in vivo is thecomplete elimination of the apoptotic cell before an inflammatoryresponse is seen. Necrosis usually causes inflammation. Though apoptosiscan be thought of as a clean and neat process, the p53 targetingtreatments do not result in apoptosis in all types of cancer cases.Though necrosis may typically cause an inflammatory response to atreatment site directed at targeting HDM-2, HDM-2-targeting treatmentsare more effective against various forms of cancer, including thosewhere p53 is not present in the cancer cells, or where p53 is in amutated or an inactive form.

Human pancreatic cancer cells, MiaPaCa-2 cells, were treated withPNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH)as well as elevation of pro-apoptotic proteins. Mutant PNC-compound(PNC-29) and HDM-2-binding domain p53 aa12-26 without MRP (PNC-26) werecontrols. PNC-29 and PNC-26 are both used as controls, as PNC-29includes a non-p53 peptide bound to the MRP, and PNC-26 includes aa12-26 of the p53 binding domain but no MRP.

Since the inventors have discovered evidence that MRP is required foranti-cancer activity, the inventors of the present invention tested“naked” p53 peptide without MRP by transfecting a plasmid that encodesp53 aa17-26 segment of PNC-28 into MiaPaCa-2 and an untransformed ratpancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy wasemployed to further elucidate anti-cancer mechanism.

The inventors acquired the following results from the above experiment.Treatment with PNC-28 does not result in the elevation of pro-apoptoticproteins found in p53-induced apoptosis, but elicits rapid release ofLDH, which is indicative of tumor cell necrosis. Accordingly, usingtransmission electron microscopy, the inventors of the present inventionobserved membrane pore formation and dose-dependent killing. In directcontrast, MiaPaCa-2 cells, that were transfected with a vectorexpressing p53 aa 17-53, as in PNC-28, underwent apoptosis, and notnecrosis, as evidenced by expression of high levels of caspases-3, 7 andannexin V with background levels of LDH.

These results suggest that PNC-28 may be effective in treating humanpancreatic cancer. More particularly, these results suggest thatcompounds having an HDM-2 binding domain which is attached to an MRP atthe carboxyl terminal end may be effective in treating human pancreaticcancer. The MRP appears responsible for the fundamental change in themechanism of action inducing rapid necrosis initiated by membrane poreformation. Cancer cell death by apoptosis was observed in the absence ofMRP. Thus, PNC-28 and compounds of similar form and function will causecancer cell necrosis by a cell membrane pore formation mechanism, ratherthan a p53 targeted treatment within the cell, which causes necrosis.

The inventors of the present invention have developed two peptides,PNC-27 and PNC-28, that contain p53 protein residues 12-26 and 17-26,respectively, attached to a MRP. Although originally conceived to blockthe binding of p53 to HDM-2 in cancer cells, thereby increasing thehalf-life of p53 preventing its ubiquitination and proteosomicdegradation, it was determined that these compounds caused cancer celldeath even in cells that lacked p53 expression (1,3). The principalsfurther observed that in cancer cells treated with these compounds,there was no increase in expression of p53-induced pro-apoptoticproteins such as caspase and Bax (1-3). Rather, these compounds inducedtumor cell necrosis as evidenced by the rapid release of lactatedehydrogenase (LDH) from treated cancer cells (2, 3).

Interestingly, fluorescent probe-labeled PNC-27 was detected at earlystages after treatment in the cell and nuclear membranes (2). Time-lapseelectron microscopy studies later revealed that both compounds inducedpore formation in the cell and nuclear membranes, consistent with thecompounds being membrane active (2). Furthermore, consistent with thisactivity, the three-dimensional structure of PNC-27 by two-dimensionalNMR was determined to be an amphipathic alpha-helix-loop-alpha-helix, astructural motif similar to that of a number of membrane-active peptides(4, 5). Importantly, the principals devised a double-fluorescent-labeledPNC-27 that contained a green fluorescent probe (fluoresceinisothiocyanate, i.e., FITC) on the amino terminal end and a redfluorescent probe (rhodamine) on the carboxyl terminal end (MRP-end).When this double-labeled PNC-27 was incubated with MIA-PaCa-2 and MCF-7cancer cells, a high density of yellow fluorescence confined to the cellmembrane after 1 hour of incubation was identified. The only manner inwhich yellow fluorescence could be obtained is if the amino and carboxylterminal ends of the compound stay together, i.e., there is no splittingof PNC-27 into HDM-2 targeting and MRP portions/components. During thistime, there is a maximal release of LDH into the incubation medium,indicating maximal cell membrane damage. Thus the full PNC-27 compoundis required for cell membrane lysis and cancer cell death.

Though these compounds induce tumor cell necrosis among a wide range ofdifferent human tumors, including TUC-3 metastatic pancreatic cancercells (6), they remarkably have no effect on the growth and viability ofa number of normal cell lines. These include rat pancreatic acinarcells, called BMRPA1 (1,3), the normal counterpart of TUC-3 cells, humanbreast epithelial (MCF-10-2A) cells (2), and cord blood-derived humanstem cells (1). These compounds also have no effect on the growth orviability of human keratinocytes and human fibroblasts. As previouslyshown, both compounds appear to induce the killing of cancer but notnormal cells by a novel membranolytic mechanism (2).

In contrast, several studies (7-12) reported p53-dependent apoptosis oftreated cancer cells when synthesized peptide sequences targeted to bindto intranuclear HDM-2 were attached to leader sequences on their aminoterminal ends. In one such study (12), twelve residues from the h (or m)dm-2 binding domain of p53 were synthesized and attached at their aminotermini to a TAT leader peptide. This peptide was found to induceapoptosis of uveal melanoma and retinoblastoma cell lines, bothcontaining wild-type p53. Although active against cell lines homozygousfor mutant p53, this peptide was not tested against p53-null cells.Interestingly, placement of the MRP on the amino terminal end of the p5317-26 peptide resulted in a marked diminution in the cytotoxicity of thecompound to cancer cells (M Kanovsky, M R Pincus & J Michl, unpublishedobservations).

Since PNC-27 and PNC-28 both contain p53 sequences involved in thebinding of p53 to HDM-2 but induce cancer cell death via membranolysisin a p53-independent manner and display a structural motif of a membraneactive component that depends on the presence of the MRP on the carboxylterminal end of the p53 sequence, the present inventors inquired as towhether MRP plays an essential role in the membranolytic activity ofthese compounds.

To investigate this question, the “naked” p53 17-26 peptide, i.e., withno MRP, was introduced into a human pancreatic cancer cell line,MiaPaCa-2, by transfecting a plasmid encoding this peptide into thesecells in which peptide expression occurs. The inventors also treatedthese cells with PNC-28. In both conditions, the inventors measuredexpression of markers for apoptosis and necrosis to explore whether thenaked peptide induces apoptosis in contrast to the same peptide linkedto MRP on its carboxyl terminal end (which induces necrosis).

PNC-28, but not Negative Control Peptide PNC-29, is Cytotoxic toMiaPaCa-2 Cells. PNC-28 was incubated with 6×10⁶ Mia-PaCa-2 cells for 5days at concentrations ranging from 12.5-75 μmol/ml. The anti-cancereffect on MiaPaCa-2 cells incubated with PNC-28 is shown in FIG. 1(A-C). FIG. 1A demonstrates untreated MiaPaCa-2 cells that are notcontact-inhibited and spindle-shaped, many becoming multinucleated.After 24 h of treatment with 75 μmol/ml PNC-28, these cells appearnecrotic demonstrated by membrane blebbing and disruption, forming cellclumps coalescing into aggregates of cellular debris (FIG. 1B). At 48 h,there was near 100 percent cell death as measured by trypan blue dyeuptake. In contrast, negative control peptide PNC-29 at 75 μmol/ml hadno effect on cellular growth, morphology, and viability (FIG. 1C). Thus,FIGS. 1A through 1C illustrate that peptide sequence that correlates top53 aa residues 17-26 (PNC-28 peptide sequence) with the MRP, as opposedto non-p53 peptide sequence with MRP, causes necrosis, resulting in merecell clumps and cellular debris when administered to the humanpancreatic cancer cell line MiaPaCa-2. Thus, the MRP causes poreformation in the membrane of cancer cells when in combination with a p53aa residue, which binds to HDM-2 in the cell membrane.

In FIG. 2 , inhibition of proliferation was obtained after only 48 h ofpeptide treatment. The effective compound dose ranged between 20 and 75μmol/ml. It should be noted that doses of PNC-28 between 20 and 75μmol/ml induced virtually 100 percent cell death; the times required forcell killing decreased as dose increased. For example, 80 μmol/mlinduced near total cell death in 48 h while 40 μmol/ml induced similarcell death in 4 days, and 20 mol/ml induced cell death in 1 week. FIG. 2illustrates that, as the dosage of compound PNC-28 increases, so toodoes the percent of cell death measured in a 48 hour period. While it ispossible to administer a dosage above 160 μmol/ml, the cancer cells willnot be necrosed at any greater of a speed. Thus, 160 μmol/ml is thedesired upper limit of the dosage for the cell sample sized used withthese experiments.

FIG. 3 summarizes the cytotoxic effects on Mia-PaCa-2 cells of PNC-28but not control compounds including PNC-29 and the “naked” p53 17-26peptide (without the MRP), PNC-26, which cannot traverse the cellmembrane since it lacks the MRP. Since PNC-29 contains the MRP, but notthe p53, sequence and has no effect on cell growth, the MRP itself doesnot induce the observed cytotoxicity. FIG. 3 illustrates that the numberof cells in the sample untreated as compared to treated with the twocontrols is relatively the same; in contrast, PNC-28 cell death ofcytotoxicity is far greater than the negative controls and control.Thus, to cause necrosis by the proposed HDM-2 mechanism, the compoundpreferably includes both an HDM-2 binding site (HDM-2 targetingcomponent) and a MRP.

As a further control, the inventors' original experiment (1) byincubating 75 mol/ml PNC-28 with untransformed BMRPA1 acinar cells (1)and with the untransformed breast epithelial cell line, MCF-10-2A (2).There was no growth inhibition or cytotoxicity found (data not shown;see refs. 1 and 2). These results suggest that PNC-28 is lethalspecifically to cancer cells and does not interfere with normal cellgrowth, as concluded in the inventors' previous studies (1,2).

Markers for Necrosis and Apoptosis in MiaPaCa-2 Cells Treated withPNC-28. In previous studies by the present inventors, it was found thatPNC-28 induced cancer cell death in a variety of human cancer cells(1,2) by inducing tumor cell necrosis rather than apoptosis (2). Thiswas manifested in baseline expressions of caspase but high levels of LDHwithin 24 h in the medium indicative of membrane lysis. Therefore, theexpression of LDH and caspase in MiaPaCa-2 cells treated with PNC-28 wasinvestigated. FIG. 4A shows that LDH activity is elevated in cellstreated with PNC-28 (condition 2) almost to the same extent as cellsthat were lysed with lysis buffer (condition 1). On the other hand, onlybaseline levels of LDH were found for cells treated with negativecontrol PNC-29. As is shown in FIG. 4A, when PNC-28 is administered tocancer cells, it exhibits similar lysis causing conditions as theadministration of a lysis buffer; whereas the PNC-29 which has no HDM-2binding domain and does not cause lysis (and exhibits an optical densitycloser to the control where no compound—or peptide—is administered).Along the Y axis, OD refers to optical density, or absorption, of LDH,the necrosis indicating factor.

The premise that PNC-28 induced tumor cell necrosis is supported byelectron micrographs of MiaPaCa-2 cells treated with this compound in astudy that is identical to the one performed on breast cancer cells bythe present inventors (2). As shown in FIG. 4B, MiaPaCa-2 cells treatedwith PNC-28 (left panel) exhibit lysis of their plasma membranes aspreviously determined for breast cancer cell lines (2), in contrast tountreated cells (right panel) that have their plasma membranes intact.This pattern is characteristic of tumor cell necrosis (2). Thus, PNC-28is able to both bind to the cell membrane and also transport at leastpart of the compound molecule through the cell membrane, which resultsin the pore formation, or lysis, shown in FIG. 4B.

In contrast, as can be seen in FIG. 4C, only baseline levels of caspasewere expressed in MiaPaCa-2 cells treated with PNC-28 and were identicalto the level expressed in cells treated with control, PNC-29. Thisfinding confirms the conclusion that PNC-28 does not induce apoptosis.Thus PNC-28 induces tumor cell necrosis in MiaPaCa-2 cells as found forother cancer cell lines (1,2). TNF-α is a necrosis inducing agent. Onthe Figure, UL refers to units luminescence, or luminescence intensity.

The results shown in FIGS. 4A-C were obtained using 25 μmol/ml PNC-28.As shown in FIG. 4D, the same results were obtained with all doses ofcompound that were used over the 20-75 μmol/ml range, i.e., earlyrelease of LDH (necrosis indicating) but only background levels ofcaspase (apoptosis indicating), suggesting that tumor cell necrosis isinduced at all concentrations of PNC-28 and that this mechanism ofinduction of cell death is not dependent on PNC-28 concentration. As isshown on FIG. 4D, at 25 umol/ml PNC-28, caspase was approximately 0.03,while LDH was approximately 0.235 OD; at 50 umol/ml PNC-28, caspase wasapproximately 0.02, while LDH was approximately 0.44 OD; at 75 umol/mlPNC-28, caspase was approximately 0.015, while LDH was approximately0.35 OD; and at 75 umol/ml PNC-29 (negative control), caspase wasapproximately 0.05, while LDH was approximately 0.01.

Transfection of MiaPaCa-2 Cells with a Plasmid that Encodes the p5317-26 Sequence. Results of Transfection of MiaPaCa-2 and BMRPA1 Cells.After 2 hours post-transfection, cell counts were performed on slidesusing light microscopy and then counted the number of cells exhibitinggreen fluorescence from GFP (Green Fluorescent Protein). On this basis,it was found that between 30 and 45 percent of the cells expressed GFPas summarized in FIG. 9 . The highest transfection rates were found forMiaPaCa-2 cells whether transfected with empty vector (EV) or p5317-26-encoding vector (p53 17-26-V).

Morphological Examination of Transfected Cells, as Visualized byInverted Light Microscopy: In the initial set of transfectionexperiments, cells were observed by light microscopy beginning 18 hpost-transfection. MiaPaCa-2 cells transfected with p53 17-26-V werevisibly hypertrophic, many showing membrane blebbing and some werenecrotic. In contrast, BMRPA1 cells transfected in the same way asMiaPaCa-2 by either EV or p53 17-26-V showed little alteration inmorphology. This observation was confirmed 90 h post-transfection, asthe cells by this time point resumed their normal polygonal epithelialcell morphology, identical to that of untreated cells.

Cell Viability Post-Transfection. FIG. 5 shows the effect oftransfection of the plasmid p53 17-26-V encoding the p53 17-26 peptideon cell viability for MiaPaCa-2 and untransformed BMRPA1 cells. As canbe seen in this FIG. 5 , within 48 h, transfection of this plasmidinduces 60 percent cell death (condition 1) while transfection of p5317-26-scrm-V control plasmid results in a much lower level of celldeath, i.e., 20 percent as shown in condition 2. This is a baselinelevel since this is the level of cell death observed for untransformedBMRPA1 cells transfected with the same control vector, condition 4. Incondition 3 of this FIG. 5 , it can be seen that expressed p53 17-26peptide has a much less pronounced effect on BMRPA1 cells, resulting inthe same baseline level of cell death seen in controlplasmid-transfected BMRPA1 cells (condition 4). Thus, expression of thecompound induces cell death in cancer, but not in untransformed, cells.

Effects of the p53 17-26 Peptide on MiaPaCa-2 Cells. MiaPaCa-2 cellsexpressing GFP that had been transfected with EV or p53 17-26-V werelysed and blotted for p53, waf^(p21), a protein that is induced by ap53-dependent pathway, and the p53 17-26 peptide itself. In theseexperiments, the DO-1 anti-p53 antibody that recognizes a determinantthat contains residues 17-26 of p53 was used (2). In addition, caspaseactivity in these cells was measured. For comparison, the same set ofexperiments was performed on Mia-PaCa-2 cells treated with 75 μmol/ml ofPNC-28. As can be seen in FIG. 6 , at 0 time (left side of FIG. 6 ,labeled “0 time”) after transfection or incubation with PNC-28, peptide,p53, waf^(p21) and caspase activity are all expressed at baselinelevels. At times when cell death was near 100 percent at 96 hr fortransfected cells, 48 hr for PNC-28-treated cells, peptide levels werefound to be high in both transfected and PNC-28-treated cells (FIG. 6 ,right side, labeled “100% cell death”). However, in the transfectedcells, it can be seen that there are elevated levels of p53, waf^(p21)and caspase activity (labeled “transfection” on the right side of thefigure) that are not observed in the PNC-28-treated cells (labeled“PNC-28” on the right side of the figure). For controls, actin wasblotted for and it was found that the levels were the same for all fourconditions in FIG. 6 (not shown). These results suggest that the p5317-26 peptide induces increased intracellular expression of p53 proteinwith consequent apoptosis of MiaPaCa-2 cells, as evidenced by theconcomitant increased expression of waf^(p21) that does not occur incells treated with PNC-28. Along the right axis, luminescence intensityis measured at 405 NM. Along the left axis, 450 NM measures the bandintensity of the Western Blot. Interestingly, when lysates fromuntransformed BMRPA1 cells transfected either with EV or with p5317-26-V were blotted, only low levels of expression of p53 were found.In addition, only a low level of expression of p53 17-26 peptide in p5317-26-V-transfected cells was found (results not shown). Since GFP wasexpressed at high levels in these cells, it appears that expressedpeptide is unstable in these untransformed cells.

Expressed p53 17-26 Peptide Induces Apoptosis, not Necrosis, ofMiaPaCa-2 Cells. Since expression of p53 and waf^(p21) was elevated incells transfected with p53 17-26-V to much higher levels than in cellstransfected with control vector, it was concluded that the peptide wasinducing apoptosis in contrast to its counterpart PNC-28 peptide asdiscussed above. Further confirmation of peptide-induced apoptosis wassought. In the early stages of apoptosis, phosphatidyl serine (PS),normally present in the inner leaflet of the bilayer membrane of intactcells, is found on the external plasma membrane of cells undergoingapoptosis. Annexin V binds PS and can be located by a probe that carriesthe red fluorescent TRITC probe. Consequently, cells that had beentransfected approx 48 h earlier were processed for staining with AnnexinV-biotin followed by streptavidin-TRITC. FIG. 7 shows the confocalmicroscopic results for MiaPaCa-2 cells transfected with control vector(upper left), showing green fluorescent cells with no red staining, andcells transfected with p53 17-26-V that show green fluorescent cellswith strong red staining for PS. On the right side of the figure are theresults for BMRPA1 control cells that have been transfected with controlvector (upper right) or p53 17-26-V (lower right). As can be seen inFIG. 7 , neither panel is positive for PS in the normal control cells.As discussed above, expression of p53 17-26 peptide is low in this cellline, possibly causing the absence of signs of apoptosis. Thus the p5317-26 peptide induces apoptosis in the cancer cell line only.

Caspase and LDH in Transfected MiaPaCa-2 Cells. To compare the resultswith those from MiaPaCa-2 cells treated with PNC-28 peptide (FIG. 4 )p53 17-26-V-transfected cells were assayed for caspase and LDH release.As shown in FIG. 8A, condition 3, caspase expression is over four-foldhigher in these treated cells than in untreated cells (condition 2) andhas the same fold-increase over that in cells treated with PNC-28 andcontrol PNC-29. In contrast, as shown in FIG. 8B, LDH release from thesetransfected cells (condition 3) is at the baseline level found foruntreated cells (condition 2) and is about five-fold lower than releasefrom cells treated with PNC-28 (condition 4). Thus, presence of theMRPin PNC-28 results in a change in the mechanism of action of the p5317-26 peptide; without MRP, the peptide induces apoptosis, while, withMRP on its carboxyl terminal end, the peptide induces tumor cellnecrosis.

Prior Evidence that the MRP in PNC-27 and PNC-28 Is Required forInduction of Tumor Cell Necrosis. The purpose of this study was todefine the role of the MRP in PNC-28 in inducing tumor cell necrosis. Inprevious studies with this MRP and its compound, PNC-27, it was foundthat both PNC-27 and PNC-28 induced tumor cell necrosis, not apoptosis,and caused necrosis even in cancer cells in which p53 protein was absent(1-3). These findings suggested that both PNC-27 and PNC-28 exertedtheir effects independently of p53 activation. They contrasted with theresults of studies in which similar p53 sequences, were attached to MRPon their amino terminal ends, and induced p53-dependent tumor cellapoptosis, not necrosis (7-11). This results from the binding of thesepeptides to HDM-2 in place of the p53 protein; these peptides are notthemselves ubiquitinated since the sites for p53 ubiquitination lieoutside this domain (16).

Further evidence that the presence of the MRP on the carboxyl terminalend of PNC-27 and 28 is essential for its induction of tumor cellnecrosis is the three-dimensional structure of PNC-27. PNC-27 was foundto have a highly amphipathic alpha-helix-loop-alpha-helix structure thatis found in membrane-active peptides (4). Disruption of this structure,as may occur by placement of the MRP on the amino terminal end of thecompound, would be expected to change the activity of the compound. Itwas found that the p53 17-26 peptide containing the MRP on its aminoterminus, called reverse or r-PNC-28, has much lower activity in cellkilling than does PNC-28 itself (M. Kanovsky, J. Michl and M. R. Pincus,unpublished observations). These findings support the conclusion thatthe MRP is critical to the activity of the compound but leaves open thequestion as to whether the “naked” p53 peptide itself can inducenecrosis or apoptosis of cancer cells.

Cells Transfected with pTracer-SV40 Plasmid Encoding p53 17-26 “Naked”Peptide Express this Peptide. To define the role of the MRPdefinitively, it was sought to determine the effects of the p53 17-26peptide itself on tumor cell growth, i.e., whether even without the MRP,it could induce tumor cell necrosis. To accomplish this goal, the p53peptide was introduced into MiaPaCa-2 cells via transfection using thepTracer-SV40 plasmid that constitutively expressed this peptide. Then,the expression of markers for apoptosis and necrosis in the transfectedcells and compared the levels of these markers with those found inMiaPaCa-2 cells treated with PNC-28 was determined.

As can be seen in FIG. 9 , transfection efficiencies were relativelyhigh. MiaPaCa-2 cells transfected with the p53 peptide-expressingplasmid expressed high levels of the p53 peptide and expressed highlevels of GFP as revealed by Western blots over this time period. By 90hours, when at least two-thirds of the cells were killed (FIG. 7 ),peptide expression decreased to barely detectable levels while GFPlevels also decreased significantly (FIG. 6 ). This phenomenon may havebeen caused by cell death and release of proteases causing peptidedegradation. On the other hand, BMRPA1 cells transfected with the samepeptide-encoding plasmid expressed much lower levels of this peptide.Since these cells expressed high levels of GFP, which is expressed underthe same promoter, it is not likely that p53 peptide was not also beingsynthesized in these cells. One possible explanation for thisobservation is the status of HDM-2. Recently, it was found by thepresent inventors that this protein is expressed at barely detectablelevels in untransformed cells, including BMRPA1 cells, but is expressedat high levels in transformed cells. If binding of the small p53decapeptide to HDM-2 blocks its degradation in cells, absence of HDM-2may make the peptide susceptible to intracellular proteases resulting inits degradation.

The p53 17-26 Peptide Induces Apoptosis of Cancer Cells. As summarizedin FIG. 6 , expression of the p53 17-26 peptide in MiaPaCa-2 cellsinduces increasing expression of p53 with concomitant increasingexpression of waf^(p21) over a time period in which cell deathincreases. These results are compatible with binding of the peptide tohdm-2, blocking the binding of p53 to this protein, resulting inprolongation of the half-life of p53. This would result in its increasedexpression in MiaPaCa-2 cells, allowing it to cause apoptosis,explaining the increasingly elevated levels of waf^(p21). As shown inFIG. 8A, caspase, a marker for p53-dependent apoptosis, is elevated toalmost five times the background level (condition 2) in p53 17-26peptide-expressing MiaPaCa-2 cells (condition 3). Likewise, it iselevated above background in cells incubated with PNC-28 (condition 4).Also, in virtually all MiaPaCa-2 cells treated with p53 17-26peptide-expressing cells, there was strong expression ofannexin-V-binding phosphatidyl serine in the membranes of transfectedMiaPaCa-2 cells, a known early phenomenon in apoptosis (15), but not inMiaPaCa-2 cells transfected with empty vector (FIG. 7 ). These resultssuggest that the naked p53 17-26 peptide causes cancer cell death byinducing apoptosis. Furthermore, the p53 peptide-expressing MiaPaCa-2cells do not release LDH in 24 h (condition 3, FIG. 8B) as would beexpected if the peptide induced tumor cell necrosis. In contrast,treatment of MiaPaCa-2 cells with PNC-28 resulted in high levels of LDH(condition 2, FIG. 4A and condition 4 in FIG. 8B) over this time coursethat began as early as several minutes after treatment, confirming thatthe peptide induces tumor cell necrosis and not apoptosis. This effectis independent of the concentration of PNC-28, suggesting that itsmechanism of action does not change in a concentration-related manner.This finding, that expression of the p53 17-26 peptide in a cancer cellline induces apoptosis, is in agreement with the results of priorstudies that showed that similar peptides from the HDM-2 binding domainof p53 likewise induce apoptosis (7-12).

Overall, experimental results set forth herein strongly suggest that thep53 17-26 peptide induces tumor cell apoptosis and not necrosis. On theother hand, presence of the MRP to the carboxyl terminal end of the p5317-26 peptide plays a critical role in changing the mechanism of cellkilling of this peptide from apoptosis to tumor cell necrosis.

The p53 17-26 Peptide Induces Apoptosis in Tumor but not Normal Cells.As shown in FIG. 5 , transfection of empty vector in MiaPaCa-2 cellscauses a low level of cell death. However, as shown in FIG. 7 ,transfection of empty vector into MiaPaCa-2 cells causes no exposure ofphosphatidyl serine as revealed by absence of annexin V bindingsuggesting that the transfection does not induce apoptosis. On the otherhand, transfection of the vector inducing expression of the p53 17-26sequence into these cells induces higher rates of cell death (FIG. 5 ).In all of these cells, as shown in FIG. 7 , there is strong annexinbinding suggesting that these cells are all undergoing apoptosis.

In contrast, transfection of the p53 peptide-expressing vector intountransformed BMRPA1 cells does not result in an increase in cell deathover the background (FIG. 7 ). Furthermore, neither transfection ofempty vector or of p53 peptide-expressing vector results in any exposureof annexin-binding phosphatidyl serine (FIG. 8 ). These findings may bedue at least partly to the low level of expression of the p53 17-26peptide. Nonetheless, the p53 peptide-encoding plasmid does not induceapoptosis in untransformed BMRPA1 cells. Thus, like PNC-27 and PNC-28,the p53 17-26 peptide appears to be selective for killing cancer but notuntransformed cells.

In summary, the MRPon the carboxyl terminal end of PNC-28 causes it toinduce tumor cell necrosis. Removal of the MRP from the p53 17-26peptide in cancer cells results likewise in cytotoxicity to these cellsexcept by apoptosis of the tumor cells. Thus presence of the MRP on thecarboxyl terminal end of p53 peptide results in a fundamental change inthe mechanism of action of the compound in causing tumor cell death.Like the full peptide, PNC-28, the naked p53 17-26 peptide appears to beselective to inducing apoptosis of cancer cells but not of untransformedcells. This may be due in part to low levels of expression of thepeptide in untransformed cells that express low levels of HDM-2 that mayshield expressed peptide from protease degradation. This finding impliesthat introduction of the naked peptide into cells can induce tumor cellapoptosis while leaving normal cells unaffected.

These results underscore the importance of elucidating the p53-hdm-2interaction in the cancer cell, whereby it is possible to gain a betterunderstanding of the manner of which this complex potentiates a possiblecross-talk between necrotic and apoptotic pathways that may lead tonovel approaches for directed therapy. As a result, such with HDM-2targeting component(s) and MRP(s) therapeutics may deliver selectivecytotoxic small molecules to cancer cells targeting pathways inducingnecrosis, apoptosis or both.

A method of selectively necrosing cells is provided. The method includesthe steps of providing a plurality cells, including at least one cancercell and at least one non-cancerous cell; and administering to the cellsa compound, including an HDM-2 targeting component and a cytotoxiccomponent, said cytotoxic component attached to said HDM-2 targetingcomponent, wherein said compound comprises a membrane-active form. Thecytotoxic component may desirably be, for example, a membrane residentpeptide (MRP), a toxin, a drug, a radionuclide, a whole antibody, anantibody fragment, and combinations thereof. The HDM-2 targetingcomponent may be, for example, a small molecule, a peptide, a protein, aglycoprotein, a whole antibody, an antibody fragment, and combinationsthereof.

One or more of the methods may optionally include the step of observing,in a cell medium, a level of LDH. A level of LDH may be an amount of LDHmeasured as omitted from a number of cells. LDH is known necrosisindicator, thus, the presence of LDH in the cell medium of cells treatedwith the compound of the present method indicates that at least some ofthe cells have undergone necrosis, cell death. Necrosis may similarly beobserved in a sample of cells by microscopically inspecting the cells todetermine whether the cell membranes are intact or have transmembranepore formation therein/thereon.

Though the compound of the present method selectively necroses cancercells, the compound has no observable effect on non-cancerous cells.Thus, the method may further comprise the step of observing for anon-response in a non-cancerous cell. The cells may be observed for poreformation, cell breakdown, and the like. However, observation and/oranalysis of the non-cancerous cells will yield no effect on thenon-cancerous cells. Similarly, this no effect may be referred to as anon-response by the non-cancerous cells to the cancer treatment(compound) administered. As such, the present method results in necrosisof cancer cells, while non-cancerous cells are unaffected.

A method of causing membranolysis in at least one cancer cell isprovided. The method of causing membranolysis in at least one cancercell further includes administering to at least one cancer cell acompound comprising an HDM-2 targeting component and a pore formingcomponent attached to said HDM-2 targeting component, wherein saidadministering step results in at least one transmembrane pore in acancer cell membrane. The pore forming component may include, forexample, any chemical moiety with a pore forming character when put intoassociation with a cell membrane, desirably, a cancer cell membrane. Thepore forming component may include, for example, a membrane residentpeptide (MRP).

Causing membranolysis in cancer cells is a desirable basis for cancertreatments. The present method may be employed to more readilyunderstand the dosing effectiveness of the cancer treatment and/orcompound. Thus, administration of multiple dosages may be completed inorder to more readily understand the upper and lower limits ofeffectiveness, if any. Further, the effectiveness of various treatmentplans, repeating administration, may be studied utilizing this method.Also, various types of cancer cells and/or pre-cancerous cells (atypicalcells) may be studied to analyze and determine varying levels ofaggressiveness, progression, treatability, and/or responsiveness todosages of the compound of the present method.

The method of causing membranolysis in at least one cancer cell mayfurther include observing membranolysis in the cancer cell by: detectingan LDH amount, performing electron microscopy, observing cellmorphology, and combinations thereof. By observing membranolysis, theeffectiveness of various dosages and, potentially, the effectivenesscombination therapies, may be better understood. The observation ofmembranolysis is linked to necrosis in the cancer cells, as thestructural integrity of the cell is no longer maintained oncetransmembrane pores trigger membranolysis. However, varying degrees ofnecrosis may be observed. This may be due, in part, to the naturalbreakdown of the compound within a sample or a subject. Further, thismay be linked to dosage, strength of the cancer cells, or duration oftreatment.

A method of treating cancer in a subject in need thereof is provided.The method includes administering to the subject in need thereof atherapeutically effective amount of a compound having an HDM-2 targetingcomponent and a membrane resident peptide (MRP), said HDM-2 targetingcomponent and said MRP having a membrane-active form. After theadministration step, optionally, the method may include determiningwhether a plurality of cancerous cells have undergone membranolysis.

The cytotoxic component may include, for example, a membrane residentpeptide, a toxin, a drug, a radionuclide, a whole antibody, an antibodyfragment, and combinations thereof. The HDM-2 targeting component isselected from the group consisting of: a small molecule, a peptide, aprotein, a glycoprotein, a whole antibody, an antibody fragment, andcombinations thereof.

A method of screening cancer treatments is provided. The method includesproviding a plurality of cancerous cells; each of said cells havingHDM-2 in said cellular membranes; administering a candidate cancertreatment to the plurality of cancer cells; and measuring the level ofLDH present in a cellular medium. LDH is a known necrosis factor, thus,the measurement of a level or amount of LDH present after administeringa candidate cancer treatment to the cancer cells with detect whether thecancer treatment was successful in causing cell death to cancer cells.

Optionally, the candidate cancer treatment may be screened to determinewhether the candidate cancer treatment has membrane active conformation.The membrane active conformation may refer to the desired shape of thecandidate cancer treatment in solution. The conformation may be directlylinked to the candidate cancer treatment's ability to colocalize withHDM-2 in the cancer cell membrane and be retained within the cancer cellmembrane, causing the formation of pores therein. Desirably, candidatecancer treatments may have a three dimensional shape or conformation inan alpha-helix-loop-alpha-helix. This is the three-dimensional shapethat has been determined by the present inventors for the PNC-27 andPNC-28 peptide-based compositions. The alpha-helix-loop-helixconformation allows the composition to advantageously interact with thecancer cell membrane.

Optionally, the candidate cancer treatment may be screened to determinethe candidate cancer treatment includes an HDM-2 targeting component. Todetermine this, it is possible to analyze the HDM-2 targeting componentto determine whether it has any affinity and or binding capability toHDM-2 in cancer cell membranes.

Optionally, the method may further include the step of observing thecancer cell membranes for an area of pore formation. Pore formation isthe mechanism by which membranolysis is caused, which results in cancercell death. Pore formation may be microscopically observed. Also,various assays may be completed, measuring known necrotic factors,including, for example, LDH.

A candidate cancer treatment which may be found to have a membraneactive form, cause membranolysis to cancer cells, have a similarconformation to PNC-27 and/or PNC-28, and have an HDM-2 binding affinitymay be termed a material with anti-cancer “activity”. Use of the term“activity” with respect to a cancer treatment with reference to theembodiments of the present invention refers to an ability to induce adesirable effect upon in vitro, ex vivo, or in vivo administration ofthe compound. Desirable effects include preventing or reducing thelikelihood (increasing the likelihood or causing) one or more of thefollowing events: binding to HDM-2 in cancer cells, insertion into thecancer cells' plasma membrane, assembly and pore foundation, transportacross the cancer cell membrane, causing membranolysis. Materials withanti-cancer “activity” may be flagged for further testing, review, andconsideration as viable cancer treatments.

Identifying drug candidates from cancer candidate treatments flagged ashaving “activity”, typically involves multiple phases. During the earlystages, compounds, preferably large libraries of compounds are screenedor tested in vitro for binding to and/or biological activity at thecancer cell membrane (with HDM-2 and/or a membrane resident componentcharacteristic). The compounds that exhibit activity (“active compounds”or “hits”) from this initial screening process are then tested through aseries of other in vitro and in vivo tests to further characterize theanti-cancer normal, non-cancerous tissue and organ protective activityof the compounds.

The in vivo tests at this phase may include tests in non-human mammalssuch as those mentioned above. If a compound meets the standards forcontinued development as a drug following in vitro and in vivo tests,the compound is typically selected for testing in humans.

A progressively smaller number of test compounds at each stage areselected for testing in the next stage. The series of tests eventuallyleads to one or a few drug candidates being selected to proceed totesting in human clinical trials. The human clinical trials may includestudies in a human suffering from a medical condition that can betreated or prevented by reducing cancer cells (inducing cancer cellnecrosis).

A method of selectively necrosing cancer cells is provided. The methodincludes providing a plurality of cells, including at least one cancercell and at least one non-cancerous cell; and contacting the pluralityof cells with an HDM-2 targeting compound which includes a membraneresident peptide (MRP), wherein the HDM-2 targeting compound colocalizesto HDM-2 present in at least one cancer cell membrane, binding to a cellmembrane of the at least one cancer cell and adopting amembrane-resident conformation within said cancer cell membrane.

The adoption of a membrane-resident conformation may further includeforming a pore in said cancer cell membrane. Optionally, the method mayinclude observing necrosis in the plurality of cancer cells, whileobserving necrosis was previously discussed. Similarly, the method mayinclude the step of optionally observing a non-result in thenon-cancerous cells, which is indicative of non-targeting of saidnon-cancerous cells by the method and its related compound.

A method of identifying cancer cells is provided. The method ofidentifying cancer cells includes providing a plurality of cells,wherein at least one of said cells is a candidate cancer cell; andadministering to the plurality of cells an HDM-2 recognition agent.

Candidate cancer cells, as used herein, may generally refer to cells,cellular samples, or tissues which may include cancer calls,pre-cancerous cells, and non-cancerous cells. The method of the presentinvention may be used to determine which of the cells, if any, withinthe candidate cancer cells are cancerous. The plurality of candidatecancer cells may be optionally observed to determine whether the HDM-2recognition agent colocalizes with at least one cell membrane.Colocalization may result in the HDM-2 recognition molecule being boundto or transported through the cell membrane, depending on the form ofthe HDM-2 recognition agent. If the HDM-2 recognition agent takes on theform of PNC-27, or a non-peptide component with an MRP attached thereto,the HDM-2 recognition agent will be taken into the cell membrane ofcancer cells, in a membrane active form. This is indicative of therecognition agent's tendency to be bound to HDM-2 in a cancer cell.

In order to better observe and determine which candidate cancer cells,if any, are cancerous, it is possible to optionally tag the HDM-2recognition agent with an observation aid. The observation aid may beattached to the HDM-2 recognition agent such that the observation aidfollows the HDM-2 recognition agent and does not interfere with anycolocalization to HDM-2 in the cancer cell membranes. The observationaid may include one or more materials, as may be desired. Theobservation aid may be, for example, a dye, a fluorescing agent, aradiopaque material, a radioactive isotope, and combinations thereof.One useful observation aid may include, for example, horseradish radishperoxidase.

Identifying at least one cancer cell may refer to identifying that theHDM-2 recognition agent has colocalized with, bound to, or otherwiseaffiliated with the surface of a cellular membrane containing HDM-2.This may be based on the premise that HDM-2 is contained in cancer cellsat roughly five times greater presence than in non-cancerous cells.

Once the HDM-2 recognition agent has tagged or affiliated to cancer cellmembranes present out of the total candidate cancer cells (orsurrounding non-cancerous tissue), it is possible to quantify andqualify the size, shape, progression, and general nature of a pluralityof cancer cells. Thus, a plurality of cancer cells may be mapped orplotted with respect to the surrounding frame of reference (in asubject, the surrounding anatomy and/or tissues), in order to betterunderstand the placement and size of the cancer cells (canceroustissue). The observation aids, including, for example, dyes, fluorescingagents, radiopaque materials, and the like, may aid in visualizing theHDM-2 recognition agent within a sample that may have a large amount ofcells present. Thus, once the HDM-2 recognition agent is administered tocells, various known visualization techniques, including filtered scopesto detect light at certain wavelengths of the electromagnetic spectrum,x-ray, catscan, and the like may be employed in order to better see andunderstand the plurality of cancerous cells. Thus, the map of the cancercells may be useful in diagnosing types of cancer, treatments forcancer, surgical removal thereof, observing the progression, monitoringfor relapse of cancer, and/or the responsiveness to treatments.

The compounds, agents, and or materials used in conjunction with one ormore of the methods of the present invention may refer to PNC-27,PNC-28, or combinations thereof, as discussed herein. Further, it shouldbe readily understood that non-peptide materials which may desirablyhave an HDM-2 affinity or binding site may be used in conjunction withthe MRP. Hybrid materials containing peptide and non-peptide components,along with wholly non-peptide materials may be used with one or more ofthe methods of the present invention. The synthesis of one or more ofthe compounds may be subsequently followed by purification, as iscommonly done in the art. The compounds synthesized are preferably inpurified form to be used as the compound and with the methods of thepresent invention. Thus, the present invention contemplates the use ofpeptide as well as non-peptide materials, and combinations thereof, tocause selective necrosis to cancer cells, in accordance with the presentinvention.

One or more of the methods referenced herein may optionally include areiteration or repeated administration step. That is, after theadministration step, it is possible to determine whether a plurality ofsubsequent cancer cells exist and remain intact. If so, it is possibleto complete one or more of the method steps for each of the methodspreviously discussed, including the administration of the compound,HDM-2 recognition agent, and the like.

The compounds, agents, and materials used in conjunction with one ormore of the aforementioned methods are desirably in a purified form.Purified form, as used herein, generally refers to material which hasbeen isolated under certain desirable conditions that reduce oreliminate unrelated materials, i.e. contaminants. Substantially freefrom contaminants generally refers to free from contaminants withinanalytical testing and administration of the material. Preferably,purified material is substantially free of contaminants is at least 50%pure, more preferably, at least 90% pure, and more preferably still atleast 99% pure. Purity can be evaluated by conventional means, e.g.chromatography, gel electrophoresis, immunoassay, composition analysis,biological assay, NMR, and other methods known in the art.

At least one cancer cell, as used herein, may similarly refer to aplurality of cancer cells. A plurality of cells may include, a sample ofcells, a tissue sample, a tumor, and/or even a subject having cancer. Atleast one cell may refer to one cell, a plurality of cells, a sample ofcells, a tissue sample, and/or even a subject. A plurality of cellsincluding at least one cancer cell and at least one non-cancerous cellmay refer to a mixture of cells in a sample, an area of tissue includingboth cancerous and non-cancerous tissues, and or a subject diagnosedwith cancer.

The term “subject”, as used herein may refer to a patient or patientpopulation diagnosed with, or at risk of developing one or more forms ofcancer. Also, as used herein, a subject may refer to a living animal,including mammals, which may be given cancer through transplantation orxenotransplanting which may be subsequently treated with the methods andcompounds of the present invention or which have developed cancer andneed veterinary treatment. Such subjects may include mammals, forexample, laboratory animals, such as mice, rats, and other rodents;monkeys, baboons, and other primates, etc. They may also includehousehold pets or other animals in need of treatments for cancer.

The terms “therapeutically effective dosage” and “effective amount”refer to an amount sufficient to kill one or more cancer cells. Atherapeutic response may be any response that a user (e.g. a clinicianwill recognize) exhibits as an effective response to the therapy,including the foregoing symptoms and surrogate clinical markers. Thus, atherapeutic response will generally be an amelioration or inhibition ofone or more symptoms of a disease or disorder, e.g. cancer.

Administering, as referred to by one or more of the methods of thepresent invention, may include contacting. The term “contacting” refersto directly or indirectly bringing the cell and the compound together inphysical proximity. The contacting may be performed in vitro or in vivo.For example, the cell may be contacted with the compound by deliveringthe compound into the cell through known techniques, such asmicroinjection into the tumor directly, injecting the compound into thebloodstream of a mammal, and incubating the cell in a medium thatincludes the compound.

Any method known to those in the art for contacting a cell, organ ortissue with a compound may be employed. Suitable methods include invitro, ex vivo, or in vivo methods. In vitro methods typically includecultured samples. For example, a cell can be placed in a reservoir(e.g., tissue culture dish), and incubated with a compound underappropriate conditions suitable for inducing necrosis in cancer cells.Suitable incubation conditions can be readily determined by thoseskilled in the art.

Ex vivo methods typically include cells, organs or tissues removed froma mammal, such as a human. The cells, organs or tissues can, forexample, be incubated with the compound under appropriate conditions.The contacted cells, organs or tissues are normally returned to thedonor, placed in a recipient, or stored for future use. Thus, thecompound is generally in a pharmaceutically acceptable carrier.

In vivo methods are typically limited to the administration of acompound, such as those described above, to a mammal, preferably ahuman. The compounds useful in the methods of the present invention areadministered to a mammal in an amount effective in necrosing cancercells for treating cancer in a mammal. The effective amount isdetermined during pre-clinical trials and clinical trials by methodsfamiliar to physicians and clinicians.

The compounds useful in the methods of the invention may also beadministered to mammals by sustained release, as is known in the art.Sustained release administration is a method of drug delivery to achievea certain level of the drug over a particular period of time. The leveltypically is measured by serum or plasma concentration.

The compounds of one or more of the aforementioned methods of thepresent invention may be administered to a human in an amount effectivein achieving its purpose. The effective amount of the compound to beadministered can be readily determined by those skilled in the art, forexample, during pre-clinical trials and clinical trials, by methodsfamiliar to physicians and clinicians. Typical daily doses includeapproximately 1 mg to 1000 mg.

An effective amount of a compound useful in the methods of the presentinvention, preferably in a pharmaceutical composition, may beadministered to a mammal in need thereof by any of a number ofwell-known methods for administering pharmaceutical compounds. Thecompound may be administered systemically or locally.

Any formulation known in the art of pharmacy is suitable foradministration of the compounds useful in the methods of the presentinvention. For oral administration, liquid or solid formulations may beused. Some examples of formulations include tablets, capsules, such asgelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like. The compounds can be mixed with a suitablepharmaceutical carrier (vehicle) or excipient as understood bypractitioners in the art. Examples of carriers and excipients includestarch, milk, sugar, certain types of clay, gelatin, lactic acid,stearic acid or salts thereof, including magnesium or calcium stearate,talc, vegetable fats or oils, gums and glycols.

Formulations of the compounds useful in the methods of the presentinventions may utilize conventional diluents, carriers, or excipientsetc., such as those known in the art to deliver the compounds. Forexample, the formulations may comprise one or more of the following: astabilizer, a surfactant, preferably a nonionic surfactant, andoptionally a salt and/or a buffering agent. The compound may bedelivered in the form of an aqueous solution, or in a lyophilized form.Similarly, salts or buffering agents may be used with the compound.

The compounds of the present invention may be administered intherapeutically effective concentrations, to be provided to a subject instandard formulations, and may include any pharmaceutically acceptableadditives, such as excipients, lubricants, diluents, flavorants,colorants, buffers, and disintegrants. Standard formulations are wellknown in the art. See, e.g. Remington's pharmaceutical Sciences, 20thedition, Mach Publishing Company, 2000. The formulation may be producedin useful dosage units for administration by any route that will permitthe compound to contact the cancer cell membranes. Exemplary routes ofadministration include oral, parenteral, transmucosal, intranasal,insulfation, or transdermal routes. Parenteral routes includeintravenous, intra-arterial, intramuscular, intradermal, subcutaneous,intraperitoneal, intraductal, intraventricular, intrathecal, andintracranial administrations.

The pharmaceutical forms suitable for injection include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. The ultimatesolution form in all cases must be sterile and fluid. Typical carriersinclude a solvent or dispersion medium containing, e.g., water bufferedaqueous solutions, i.e., biocompatible buffers, ethanol, polyols such asglycerol, propylene glycol, polyethylene glycol, suitable mixturesthereof, surfactants or vegetable oils. Sterilization may beaccomplished utilizing any art-recognized technique, including but notlimited to filtration or addition of antibacterial or antifungal agents.

The compounds of the present invention may be administered as a solid orliquid oral dosage form, e.g. tablet, capsule, or liquid preparation.The compounds may also be administered by injection, as a bolusinjection or as a continuous infusion. The compounds may also beadministered as a depot preparation, as by implantation or byintramuscular injection.

The compounds, agents, and materials referenced in the present inventionmay be in a “pharmaceutically acceptable carrier”. A pharmaceuticallyacceptable carrier includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic agents and thelike. The use of such media and agents are well-known in the art. Thephase ‘pharmaceutically acceptable’ refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce unwanted reactions when administered to a subject, particularlyhumans. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term carrier refers to a diluent, adjuvant, excipient orvehicle with which he compounds may be administered to facilitatedelivery. Such pharmaceutical carriers can be sterile liquids, such aswater and oils, or organic compounds. Water or aqueous solution salinesolutions, and aqueous dextrose and glycerol solutions are preferablyemployed as carriers, particularly as injectable solutions.

The synthetic peptides which may include the compounds, agents, andmaterials used with the present methods of the present invention may besynthesized by a number of known techniques. For example, the peptidesmay be prepared using the solid-phase technique initially described byMerrifield (1963) in J. Am. Chem. Soc. 85:2149-2154. Other peptidesynthesis techniques may be found in M. Bodanszky et al. PeptideSynthesis, John Wiley and Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques may be found in J. Stuart and J. S. Young, SolidPhase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill.,(1984). Peptides may also be synthesized by solid phase or solutionmethods as described in The Proteins, Vol. II, 3d Ed., Neurath, H. etal., Eds., pp. 105-237, Academic Press, New York, N.Y. (1976).Appropriate protective groups for use in different peptide syntheses aredescribed in the texts listed above as well as in J. F. W. McOmie,Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y.(1973). The peptides of the present invention may also be prepared bychemical or enzymatic cleavage from larger portions of the p53 proteinor from the full length p53 protein. Likewise, membrane-residentsequences for use in the synthetic peptides of the present invention maybe prepared by chemical or enzymatic cleavage from larger portions orthe full length proteins from which such leader sequences are derived.

Additionally, the peptides of the present invention may also be preparedby recombinant DNA techniques. For most amino acids used to buildproteins, more than one coding nucleotide triplet (codon) can code for aparticular amino acid residue. This property of the genetic code isknown as redundancy. Therefore, a number of different nucleotidesequences may code for a particular subject peptide selectively lethalto malignant and transformed mammalian cells. The present invention alsocontemplates a deoxyribonucleic acid (DNA) molecule that defines a genecoding for, i.e., capable of expressing a subject peptide or a chimericpeptide from which a peptide of the present invention may beenzymatically or chemically cleaved.

Thus, the embodiments of the present invention use the proposedmechanism of interaction between HDM-2 and the compounds of the presentinvention. By incorporating a peptide sequence that shares certain p53aa residues into the compound, the inventors are promoting the compoundto bind to HDM-2 in the cancer cell membrane. Further, by combining theHDM-2 targeting component with an MRP, as the compound is transportedover the cancer cell membrane, the binding of the compound to the cellcauses membranolysis of the cancer cell membrane. This, in turn resultsin cell death through necrosis. The inventors of the present inventionhave thus invented cancer treatments that kill cancer cells, even whenmixed with healthy cells. The methods of the present invention may beused to selectively kill cancer cells, thus creating a pinpointedtreatment in a cell sample, tissue sample, or even within a patient'sbody. The methods of the present invention thus target HDM-2 in thecancer cell membrane; rather than p53. Thus, the HDM-2 targetingtreatments of the present invention are applicable to all cancer cells,including those that have no p53 present, or may have p53 in an inactive(mutated) form.

It should be readily understood and appreciated that each of theelements and features of the present invention discussed with oneembodiment may be similarly employed with other embodiments disclosedherein, and this discussion is by no means deemed limiting to thevarious additional permutations that may be employed, for example, withthe methods presented herein.

As the methods of treatment do not cause cell death of normal cells,these methods of treatment are focused on the cancer cells, irrespectiveof the mode of administration to the cell sample. Thus, these methods oftreatment may be used for tumors or cancers that are widespread,inoperable, or otherwise not effectively treated with conventional meansor combination therapies.

Examples Materials and Methods

Peptides. The following peptides were synthesized by solid phase methodsand were purified to >95% purity (Biopeptides Corp, La Jolla, Calif.):PNC-27 containing residues 12-26 (PPLSQETFSDLWKLL) (SEQ ID NO: 8) fromthe hdm-2 binding domain of p53 and PNC-28 (ETFSDLWKLL) (SEQ ID NO: 32)containing residues 17-26 from the hdm-2 binding domain of p53, bothattached on their carboxyl terminal ends to thetransmembrane-penetrating sequence which is related to the reverseomersequence of the antennapedia sequence, KKWKMRRNQFWVKVQRG (SEQ ID NO: 1),also called MRP; the control peptide PNC-26, containing only residues12-26 of p53 and no MRP; the control peptide PNC-29, an unrelatedpeptide from cytochrome P450 (also called X13) (bold) attached to MRP(italics), whose sequence is MPFSTGKRIMLGEKKWKMRRNQFWVKVQRG (SEQ ID NO:4); and PNC-7, a peptide from the ras-p21 protein containing ras-p21residues 35-47 (TIEDSYRKQVVID) (SEQ ID NO: 7) attached to the MRP havingSEQ ID NO: 1 on its carboxyl terminal end. In addition, afluorescent-labeled form of PNC-27 was synthesized, i.e., PNC-27 peptideconjugated to the fluorescent dye, fluorescein isothiocyanate (FITC) onits amino terminal end (Biopeptides Corp.).

Cells. MiaPaCa-2 cells (human pancreatic cancer cells) were obtainedfrom the American Type Culture Collection (ATCC) (Manassas, Va.) andwere cultured in DMEM supplemented with 10 percent bovine fetal serumand penicillin/streptomycin [100 U/100 ug/ml] as recommended by theATCC. BMRPA1 cells (untransformed rat pancreatic acinar cells) werecultured as described previously (1).

Methods

Preparation of Plasmids. DNA encoding the human p53 amino acid residues17-26 sequence, corresponding to the p53 sequence in PNC-28, was clonedinto the mammalian pTracer-SV40 (green-fluorescent protein[GFP]-expressing) expression vector downstream to the SV40 promoter.This vector constitutively expresses a cloned gene (Invitrogen,Carlsbad, Calif.). Also included in the vector is another expressioncassette which is linked in tandem to the SV40-p53 17-26-expressingunit. The second expression cassette contains a CMV promoter driving theexpression of the GFP-Zeocin resistance gene fusion protein. The vectorwas used to transform TOP10F′ chemically competent E. coli following theHanahan Method of transformation (13), and plated on Zeocin-containingagar plates for overnight growth. Eight colonies were then used toinoculate cultures in Low Salt Luria Broth (1% bacterial tryptone, 0.5%yeast extract, 0.5% NaCl, and 25 μg/ml Zeocin). Cultures were grownunder constant shaking at 200 rpm for 16 h in a 37° C. incubator, andplasmids were then extracted using a Qiagen Spin Miniprep Kit.

The construct sense and anti-sense strands of the cDNA encoding the p5317-26 sequence (Invitrogen, Carlsbad, Calif.) were synthesized. Thesense strand sequence was 5′-AGTCGAATTCGCCACCATGGAAACATTTTCAGACCTATGGAAACTACTTTGAGC GGCCGCAGTC-3′) (SEQ ID NO: 31).Underlined EcoRI and NotI sites are located in 5′ and 3′ ends of thecDNA, respectively. Start and stop codons are in italics. The p53 17-26coding sequence is in bold letters. For maximum protein translation intransfected cell lines, the start codon was placed within a Kozaksequence, i.e., GCCACCATGG (SEQ ID NO: 30) (with ATG being the startcodon), which is the optimal context for initiation of translation invertebrate mRNA (13). The strands (250 nmol/ml) were annealed inannealing buffer by heating to 95° C., and then cooling to roomtemperature. The annealed double stranded p53 17-26-encoding cDNA wasthen digested with NotI and EcoRI simultaneously. A total of 20 ug ofpTracer-SV40 was digested with 60 units of NotI and 60 units of EcoRI.Double-digested pTracer-SV40 and p53 17-26-encoding cDNA were thenelectrophoresed through 0.8% and 2.5% agarose gel, respectively. Gelbands containing DNA of appropriate size were excised, and DNA contentwas extracted using the NucleoTrap Gel Extraction kit (ClonTech,Mountain View, Calif.). Purified vector and cDNA were ligated with T4ligase (12 hr, 4° C.) (New England Biolab, Ipswich, Mass.). Two ul ofligation reaction was then dispensed into a vial containing 50 ul OneShot TOP10F′ competent E. coli (Invitrogen), and the reaction mixturewas incubated on ice for 10 min, heat-shocked to 42° C. (30 sec) andincubated on ice for another 2 min. A total of 250 ul SOC medium(Invitrogen) was then added to the cells which were then shaken at 37°C. (1 hr). This transformation reaction was then diluted 1:100 or 1:10using SOC medium. A total of 50 ul of each was spread on LB platescontaining 12.5 μmol/ml ampicillin that were incubated overnight at 37°C. Eight colonies were randomly chosen to inoculate eight 5 ml overnightLB cultures in the presence of 12.5 μmol/ml ampicillin Plasmidsextracted from each liquid culture were analyzed by automated DNAsequencing using the fluorescence-based dideoxy chain terminationreaction (Genewiz, North Brunswick, N.J.). It was found that they allcontained the correct p53 17-26 cDNA reading frame associated with astop codon and a start codon embedded in the Kozak sequence. Thisconstruct plasmid is termed p53 17-26-V ([expression] vector).

Precisely the same procedure was followed for preparation of a plasmidencoding a scrambled p53 peptide sequence (residues 12-26) as follows:

(SEQ ID NO: 31) 5′-AGTCGAATTCGCCACCATG TGGGACCTGACACTACCCAAACAGCTTCTACCTTCA AGTTTGAA TGA GCGGCCGCAGTC-3′,with start, stop codons, and restriction enzyme sites denoted as above.This plasmid construct is called p53-12-26-scrm-V

Transfection into Cancer Cells. Transfection of p53 17-26-V plasmid andthe two control plasmids, one, the p53 12-26-scrm-V vector and thesecond vector encoding GFP only, called EV (empty vector) into MiaPaCa-2and untransformed BMRPA1 cells was completed. These cells were evaluatedfor: viability and expression of: p53 protein, p53 17-26 peptide,caspase, annexin binding to phosphatidyl serine and LDH. Twenty fourhours prior to transfection 5×10⁵ cells were seeded in antibiotic-freemedium into each well in a six-well tissue culture dish (TCD) andallowed to adhere overnight. To three wells, 0.8 ug of p53 17-26-Vplasmid were added. To the other three wells, 0.8 ug of empty vector orp53-12-26-scrm-V vector encoding control peptide were added. To each ofthese wells, Lipofectamine 2000 transfection agent (Qiagen) was addedsuch that the ratio of plasmid DNA (in ug) to Lipofectamine 2000 (in ul)was 1:2 (14). This ratio was determined in preliminary experimentsdescribed in the next paragraph. Transfections were performed in serum-and antibiotic-free culture medium at 37° C. for 4 h at which time theincubation was continued in complete medium, containing 10% fetal bovineserum (FBS) and penicillin and streptomycin (100 U/100 μg/ml). Afteranother 4-5 h, the cells were washed followed by re-incubation in freshcomplete medium. Transfection efficiency was measured by examining thefrequency of GFP expressing cells in the total cell population 12 hpost-transfection using a Zeiss LSM 410 Confocal Laser ScanningMicroscope.

For each cell line the effective ratio of DNA:Lipofectamine 2000 reagentwas studied and was verified in preliminary experiments using acheckerboard assay. In these experiments, transfection efficiency wasestablished by titration of different concentrations of DNA in thepresence of increasing concentrations of Lipofectamine 2000 on cellsthat had been seeded onto glass coverslips. After transfection theGFP-positive (GFP+) cells on the coverslips were quantitated by countingunder a UV Light Zeiss Epifluorescence Microscope in 3-5 consecutivefields counting 200-400 cells. These preliminary experiments helped toestablish the cell density, the amount of DNA and the DNA:Lipofectamine2000 ratio to be used, and the time for transfection to proceed.

Expression of the p53 17-26 Peptide. For protein analyses, and detectionof apoptosis, 2×10⁶ cells were seeded into 10 cm diameter TCDs andtransfected with DNA:Lipofectamine 2000 proportionally adjusted to theincreased area. When the cell density reached 90-100%, the cells of theexperimental and sham-transfected group were detached using trypsin andplated into four new TCD's in which they were allowed to grow incomplete medium. At defined time points cells were released fromadherence with 10 mM EDTA in PBS and were lysed in lysing buffer [1%Triton X-100 in 0.05 M Tris-HCl (pH 8.0), 0.15 mM NaCl, 0.02% Na azide,0.01 mg/ml phenylmethylsulfonylfluoride (PMSF), and 0.001 mg/mlAprotinin]. Protein equivalents of 10⁶ cells, i.e., —30 μg/lane, werethen subjected to SDS-PAGE using 10% Tris-HCl gels and, in someexperiments, 16% Tricine Peptide Gels (Biorad, Hercules, Calif.) todetect PNC-28 (˜3104 Da) and p53 17-26 (˜1500 Da). The separatedproteins were then electrophoretically transferred to nitrocellulosemembranes followed by immunoblotting with the mAb DO-1 to p53 AAresidues 11-25, and with mAb B-2 to GFP (each at 1 μg-2.5 μg/ml blottingbuffer), respectively (2). After washing non-reacted mAbs from themembranes, the membranes were incubated (1 h) with a secondenzyme-labeled antibody from the ECL chemiluminescence kit (Amersham,Piscataway, N.J.) to detect the presence of p53 and p53 17-26 peptide.In preliminary experiments, it was noted that identification of p53protein was easily possible within 30-90 sec of exposure while clearidentifiable binding of mAbDO-1 to p53 17-26 peptide took much longertime. The membrane was therefore cut across the 17 kDa marker(kaleidoscope's polypeptide standard), to allow for the differentialexposures. In addition, a time course of GFP expression was performed inboth MiaPaCa-2 and BMRPA1 cells that established that, during the timeperiod 48-96 h post-transfection, the cells showed the highest levels ofGFP expression. Semi-quantitation of immunoblotting results wasperformed by measuring luminosity of bands in a single scanned developedx-ray film, using the histogram option of Adobe Photoshop 5.5.Background was ascertained by measuring average luminosity of 5 areas ofthe film outside the blotting region. Opacity of each band wascalculated by the equation, Opacity=255-Luminosity-background (15).

Incubation of MiaPaCa-2 Cells with PNC-28. Duplicate sets of 6×10⁶MiaPaCa-2 cells were incubated with different concentrations of PNC-28,i.e., 5, 10, 20, 40, 80 and 160 mmol/ml. Duplicate control experimentswere also performed in which 6×10⁶ MiaPaCa-2 cells were incubated withthe control, PNC-29, present at a concentration of 75 μmol/ml. Allincubations were carried out using a protocol identical to thatdescribed in ref. 1 (1). After the cells had been allowed to adhere tothe tissue culture dish (TCD) for 24 hours the medium was removed fromeach TCD, and new medium containing the same or no peptide concentrationwas added. Medium from each TCD was removed every 24 h, and fresh mediumwith its respective peptide at the appropriate concentration was added.Cells were inspected daily for changes in cell growth, morphology, andviability. At the end of each day over a five-day period, duplicate cellcounts were performed for each incubation using the trypan blueexclusion method. In addition, cell viability was also determined by3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium bromide (MTT) assayaccording to the manufacturers' instructions (Promega Corporation,Madison, Wis., USA).

Incubation of Peptides with BMRPA1 Cells. These cells are untransformedrat pancreatic acinar cells (1). Duplicate 5-day incubations wereperformed on 6×10⁶ cells in three circumstances: with no peptide, withPNC-28 at 75 μmol/ml, and with PNC-29 at 75 μmol/ml. Cells were followedfor viability and morphology over this time period. At the end of 5days, cell counts were performed using the trypan blue exclusion method.

Immunocytochemistry for Annexin V-Binding to Phosphatidyl Serine. Todetermine whether any of the transfected plasmids induced apoptosis, thecells were evaluated to determine whether the cells containedphosphatidyl serine in the inner cell membrane, identified as binding toannexin-V, as a marker for apoptosis (15). Cells (5×10⁵) were seeded in6-well TCDs 24 h prior to transfection in antibiotic-free medium. Cellswere then either transfected with p53 17-26-V, p53 12-26-scrm-V, EV orwere left untreated. At predetermined time-points post-transfection, thecells were released using 0.5× Trypsin-EDTA, collected and processed asdescribed in the manufacturer's instructions of the Annexin V-BiotinApoptosis Detection Kit (CalBioChem, La Jolla, Calif.). The stainedcells were resuspended in antifade (Molecular Probes, OR), mounted onglass slides under a glass coverslip and evaluated for red (TRITC) andgreen (GFP) fluorescence using confocal microscopy as described above.

Evaluation of Cells Treated with PNC-28 for Caspase as a Marker forApoptosis and LDH Release as a Marker for Necrosis. Cells from cultureplates at 18, 44, 66 and 90 h time points were lysed in situ in celllysis buffer [1% Triton X-100 in 0.05 M Tris-HCl (pH 8.0), 0.15 mM NaCl,0.02% Na azide, 0.1 mg/ml phenylmethylsulfonylfluoride (PMSF), and 0.001mg/ml Aprotinin]. Lysates were subjected to 10% SDS-PAGE followed byelectrotransfer to nitrocellulose and immunoblotting with antibodies toGFP and p53 (Santa Cruz Biotechnology, Santa Cruz, Calif.).Antibody-labeled proteins were identified by chemiluminescence using ECLmethodology (Amersham)(1). Assays for elevated caspase expression wereperformed using the Clontech (Palo Alto, Calif.) for caspase (CPP32)activity (2). As a positive control for the caspase activity assay,Mia-PaCa-2 cells were incubated with tumor necrosis factor (TNF) (Sigma,St. Louis, Mo.) at a concentration of 10 ng/ml for 24 h. In addition, todetect if significant cell necrosis occurred, the CytoTox96 assay wasused (Promega, Madison, Wis.) for LDH released into the cell culturemedium as performed on several breast cancer cell lines (2).

Electron Micropscopy of MiaPaCa-2 Cells Treated with PNC-28. Time-lapseelectron microscopy (EM) was used to examine the ultrastructuralfeatures of cell death. MiaPaCa-2 cells were grown for 24 h on Thermanoxcover slips (Lux Scientific), and then treated with 25 μmol of PNC-28for 1 and 15 min, along with a corresponding control group withoutpeptide. The cells were washed with PBS solution and then fixed with2.5% gluteraldehyde-PBS. The fixed cultures were rinsed in a 0.1 Mphosphate buffer (pH 7.3), post fixed in 2% (0.08 m) osmiumtetroxide-PBS (pH 7.3), dehydrated in a graded series of ethanol andpropylene oxide and embedded in Epon 812. Sections were cut at 700 Å,stained with uranyl acetate and lead citrate and examined with a JeolJEM 1010 Electron Microscope.

Blotting of Mia-PaCa-2 Cell Lysates for p53 and waf^(p21), a Target forActivated p53. Cell lysates were prepared as described in the precedingparagraph and were subjected to immunoblotting with either DO-1 antibodydescribed in the section above for expression of the p53 17-26 peptide,a (Ab-6) monoclonal anti-p53 antibody (Calbiochem,) or with polyclonalanti-waf^(p21) antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)(1:2000 dilution) using a procedure identical to that described in thesame section above. For controls, for actin was blotted for, usinganti-actin polyclonal antibody (Santa Cruz Biotechnology).

Statistical Analysis. Analysis of growth inhibition and markers fornecrosis and apoptosis were analyzed by a two-tailed Mann-Whitneynonparametric test or a two-tailed Student T-test where appropriate. AP-value of less than 0.05 was considered significant.

Various changes and modifications can be made in the present invention.It is intended that all such changes and modifications come within thescope of the invention as set forth in the following claims.

Incorporate herein by reference in its entirety is the Sequence Listingfor the application. The Sequence listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing_txt”,created on Aug. 27, 2014. The sequence_listing.txt file is 9.33 kb insize.

What is claimed is:
 1. A method of selectively necrosing cells, comprising: providing a plurality cells, including at least one cancer cell and at least one non-cancerous cell; administering to the cells a compound, including an HDM-2 targeting component and a cytotoxic component, said cytotoxic component attached to said HDM-2 targeting component, wherein said compound comprises a membrane-active form.
 2. The method of claim 1, wherein the cytotoxic component is selected from the group consisting of: a membrane resident peptide, a toxin, a drug, a radionuclide, a whole antibody, an antibody fragment, and combinations thereof.
 3. The method of claim 1, wherein the HDM-2 targeting component is selected from the group consisting of: a small molecule, a peptide, a protein, a glycoprotein, a whole antibody, an antibody fragment, and combinations thereof.
 4. The method of claim 1, further comprising the step of observing in a cell medium a level of LDH.
 5. The method of claim 1, further comprising the step of observing necrosis in the cancer cells.
 6. The method of claim 1, further comprising the step of observing a non-response in the non-cancerous cell, wherein the non-response indicates the non-cancerous cell is unaffected.
 7. The method of claim 1, wherein the administering step further comprises administering a PNC-27, a PNC-28 peptide, or combinations thereof.
 8. A method of causing membranolysis in at least one cancer cell, comprising: administering to at least one cancer cell a compound comprising an HDM-2 targeting component and a pore-forming component attached to said HDM-2 targeting component, wherein said administering step results in at least one transmembrane pore in a cancer cell membrane.
 9. The method of claim 8, further wherein the administering step further comprises administering said compound at a dosage.
 10. The method of claim 8, further comprising the step of observing membranolysis in the cancer cell by: detecting an LDH amount, performing electron microscopy, observing cell morphology, and combinations thereof.
 11. The method of claim 8, further comprising the step of observing necrosis of the cancer cell.
 12. A method of treating cancer in a subject in need thereof, comprising: administering to the subject in need thereof a therapeutically effective amount of a compound having an HDM-2 targeting component and a cytotoxic component, said HDM-2 targeting component and said cytotoxic component having a membrane-active form.
 13. The method of claim 12, wherein the cytotoxic component is selected from the group consisting of: a membrane resident peptide, a toxin, a drug, a radionuclide, a whole antibody, an antibody fragment, and combinations thereof.
 14. The method of claim 12, wherein the HDM-2 targeting component is selected from the group consisting of: a small molecule, a peptide, a protein, a glycoprotein, a whole antibody, an antibody fragment, and combinations thereof.
 15. The method of claim 12 further comprising the step of determining whether a plurality of cancerous cells have undergone membranolysis.
 16. The method of claim 12, further comprising the step of, after the administering step, determining whether a plurality subsequent cancer cells exists.
 17. A method of screening cancer treatments, comprising: providing a plurality of cancerous cells; each of said cells having HDM-2 in said cellular membranes; administering a candidate cancer treatment to the plurality of cancer cells; and measuring the level of LDH present in a cellular medium.
 18. The method of claim 17, further comprising the step of determining whether the candidate cancer treatment has membrane active conformation.
 19. The method of claim 17, further comprising the step of determining whether the candidate cancer treatment includes an HDM-2 targeting component.
 20. The method of claim 17, further comprising the step of observing the cancer cell membranes for an area of pore formation.
 21. A method of selectively necrosing cancer cells, comprising: providing a plurality of cells, including at least one cancer cell and at least one non-cancerous cell; and contacting the plurality of cells with an HDM-2 targeting compound which includes a membrane resident peptide (MRP), wherein the HDM-2 targeting compound colocalizes to HDM-2 present in at least one cancer cell membrane, binding to a cell membrane of the at least one cancer cell and adopting a membrane-resident conformation within said cancer cell membrane.
 22. The method of claim 21, wherein adopting the membrane-resident conformation further includes forming a pore in said cancer cell membrane.
 23. The method of claim 21, comprising observing necrosis in the plurality of cancer cells.
 24. The method of claim 21, comprising observing a non-result in the non-cancerous cells, indicative of non-targeting of said non-cancerous cells.
 25. A method of identifying cancer cells, comprising: providing a plurality of cells, wherein at least one of said cells is a candidate cancer cell; and administering to the plurality of cells an HDM-2 recognition agent.
 26. The method of claim 25, further comprising of the step of observing the plurality of cells for the HDM-2 recognition agent colocalization with at least one cell membrane.
 27. The method of claim 25, wherein said HDM-2 recognition agent further comprises an HDM-2 targeting component tagged with an observation aid, said observation aid selected from the group consisting of: a dye, an enzyme, a fluorescing agent, a radiopaque material, a radioactive isotope and combinations thereof.
 28. The method of claim 25, further comprising indentifying at least one cancer cell.
 29. The method of claim 25, further comprising mapping a plurality of cancer cells. 